There is at present a huge demand for recombinant proteins in research and therapy, and it is anticipated that this demand will further expand during the next 20 years (Chu and Robinson, 2001). Amongst the most dominant proteins produced are growth factors, monoclonal antibodies, hormones and blood coagulation factors that are mainly used for clinical and pharmaceutical purposes. Specific examples of protein-based treatments are the therapy of cancer with monoclonal antibodies like bevacizumab (Avastin™) directed against vascular endothelial growth factor (VEGF) to inhibit angiogenesis, or the therapy of multiple sclerosis with interferon beta-1a (Avonex™) (Emmanouilides et al, 2004; Serarslan et al, 2008).
Currently the increasing demand for therapeutic proteins poses a significant challenge. The availability of proteins produced from natural resources is strongly limited and thus high-yield production of recombinant proteins is imperative. This is a complicated task, starting with the choice of an appropriate host organism. Comparatively simple proteins like insulin or bovine growth hormone (BGH) can be made in prokaryotic cell systems (Kwaks et al, 2006). For more complex molecules like monoclonal antibodies (mAbs) and other glycosylated proteins, eukaryotic cells are indispensable. Their properties of being able to perform correct post-translational modifications and carry out accurate protein folding and assembly are important to prevent any immunogenic effect in humans.
The production of recombinant proteins using eukaryotic cells is extremely expensive and inefficient in comparison to the production with prokaryotic cells. Nevertheless, more than 60% of all therapeutic proteins are produced in mammalian cells like Chinese Hamster Ovary (CHO) cells, Mouse Myeloma (NS0) cells, Human Embryonic Kidney (HEK-293) cells or Human Retina derived cells (PER.C6) (Ozanne, 2008; Wurm, 2004). This explains the considerable interest in developing new technologies to improve mammalian cell culture production processes. The optimisation can occur at various levels, e.g. improvement in the culture medium composition and process control as well as through vector and host cell line engineering. Regarding the improvement of the production cell lines, a post-transcriptional approach focusing on specific targeting elements in messenger RNA (mRNA) and their effect on the enhancement of protein synthesis and secretion (Knappskog et al, 2007; Stern et al, 2007a,b; Tröβe et al, 2007) is an important new area not yet fully exploited.
The mRNA targeting elements comprise the signal sequence (SS) preceding the coding sequence (cds) of a protein of interest and the 5′ and 3′ untranslated regions (UTRs). Partridge et al (1999) observed a competition between a selected SS and 3′UTR in mediating mRNA targeting to distinct polysomes. This illustrated the importance of choosing a “correct” pair of SS and 3′UTR when designing a secretion cassette. The SS encodes the signal peptide (SP), a short, cleavable polypeptide located at the N-terminus of secreted and membrane proteins, which directs protein synthesis to the membrane of the rough endoplasmic reticulum (RER), a prerequisite to attain secretion of a protein. Knappskog et al (2007) investigated the effect of different SPs derived from mammalian secretory proteins on the synthesis/secretion level of a reporter protein. Unexpectedly, the SPs derived from human albumin and interleukin-2 proved to be far inferior to that derived from the luciferase of the marine copepod Gaussia princeps. The results clearly demonstrated that the choice of SS to be used in a secretion cassette is imperative when aiming at improving protein production. Further experiments showed that SPs derived from a wide variety of sources (marine, mammalian, prokaryotic) varied greatly in their ability to promote high level protein production (Stern et al, 2007a,b; Tröβe et al, 2007). Furthermore, it has become quite clear that substantial amounts of mRNA coding for a recombinant protein can be present in transfected cells without this necessarily leading to a high level of secreted products (Knappskog et al, 2007; Tröβe et al, 2007). Thus the common strategy of merely increasing mRNA levels of a protein of interest may not be a fruitful approach.